ABSTRACT The rapid emergence of antifungal drug resistance in Candida species poses a severe antimicrobial threat worldwide. Azole antifungals, especially fluconazole, are widely used for treating Candida infections, however, azoles induce genome changes in diverse Candida species and acquired resistance can rapidly emerge. Despite this, we lack a comprehensive understanding of the molecular events that drive the acquisition of azole resistance in real time. Previously, we identified that 50% of fluconazole resistant Candida albicans clinical isolates are aneuploid and some of these aneuploidies cause pan-azole resistance. More recently, aneuploidy- acquired fluconazole resistance has been observed in many diverse human fungal pathogens, yet despite the frequency of these aneuploid events, the mechanism causing them is not known. We have developed cutting- edge multidisciplinary approaches that uniquely position us to define the rate and mechanisms of acquired azole resistance in a rigorous and reproducible way. These approaches include controlled in vitro and in vivo evolution, comprehensive comparative genomics and bioinformatics techniques, and extensive molecular genetic approaches. The aims of this application take a new approach to identifying the mechanisms driving antifungal drug resistance. In aim 1, we will determine the impact of azole stress on the rate and dynamics of acquired azole resistance. This will simultaneously enable us to identify novel and recurrent mechanisms of azole resistance, aneuploidy-derived resistance mechanisms, and the extent to which these mutations cause pan- azole and multi-drug resistance. In aim 2, the frequency of segmental chromosome aneuploidies known to cause resistance across diverse clinical isolates will be determined both in vitro and in vivo. Additionally, the impact of repetitive DNA sequences and DNA repair proteins on the formation of segmental aneuploidies will be determined. Our work is significant because it will identify the frequency, order and trajectory of mutations as they arise in a population and how these mutations cause antifungal drug resistance. Together the outcomes from these studies will identify the mechanisms that underlie how C. albicans acquires resistance and can pave the way for developing therapeutics to reduce the significant morbidity and mortality caused by diverse antifungal drug resistant Candida species.